The 600 or so proteases encoded in the human genome are involved in a diversity of biological processes. Some function as nonspecific degradative enzymes associated with protein catabolism, indiscriminately and exhaustively cleaving many protein substrates at many sites. In contrast, several others function as selective post-translational modifiers, cleaving a limited set of protein substrates, usually at only one or a few sites. Apoptosis is an important example of a biological process regulated by widespread but specific intracellular proteolysis, predominantly carried out by the caspase family of proteases. This genetically programmed and non-inflammatory form of cell death is a central component of homeostasis, tissue turnover, and development. Since apoptotic turnover of cells lies in direct opposition to the uncontrolled growth of tumor cells, a strong link also exists between apoptosis and cancer. Indeed, the terminal cellular effect of most chemotherapeutic compounds is induction of apoptosis (Kaufmann et al., Exp Cell Res, 2000, 256, 42-9).
The widespread intracellular proteolysis that is a hallmark of apoptosis is predominantly mediated by a family of aspartate-specific proteases termed caspases (Taylor et al., Nat Rev Mol Cell Biol, 2008, 9, 231-41). Apoptosis can be induced by extracellular death ligands, such as Fas ligand, TNF-α, or TRAIL, via the extrinsic pathway to activate caspase-8. It can also be induced by agents such as cytotoxic compounds, radiation, and other environmental stresses via the intrinsic pathway with release of proapoptotic factors from mitochondria to activate caspase-9. Initiator caspases-8 and -9 in turn activate downstream executioner caspases, among them caspases-3 and -7. Caspases then catalyze the inactivation of a multitude of prosurvival/antiapoptotic proteins and activation of antisurvival/proapoptotic proteins. The combined proteolytic events culminate in apoptotic cell death and clearance by phagocytes.
As a specific illustration, after receiving a cell death signal, apoptotic cells execute a cellular program that results in widespread and dramatic cellular changes that can include: (1) cell shrinkage and rounding due to the breakdown of the proteinaceous cytoskeleton; (2) the appearance of a dense cytoplasm and tight packing of cell organelles; (3) chromatin condensation into compact patches against the nuclear envelope; (4) discontinuity of the nuclear envelope and DNA fragmentation; (5) breakdown of the nucleus into several discrete chromatin bodies or nucleosomal units due to the degradation of DNA; (6) blebbing of the cell membrane into irregular buds. Near the conclusion of the apoptotic program, the cell breaks apart into several vesicles called apoptotic bodies, which are then typically phagocytosed.
Because the study of apoptotic pathways has ramifications for development of therapies for treatment of cancer, there is significant interest in gaining a better understanding of caspase proteolysis during apoptosis. For example, identification of new targets of proteolysis in apoptosis can lead to discovery of prosurvival/antiapoptotic factors, which can in turn serve as novel targets for cancer chemotherapy. A number of caspase substrates are active or established drug targets for treating cancer, including topoisomerases I and II, androgen receptor, thymidylate synthase, Bcl-2, IAPs, Mdm2 or Hdm2, PARP, HSP90, HDACs, the proteasome, Akt, MEK, Abl, EGFR, HER2, and VEGF, to name a few.
Products of caspase proteolysis may also serve as useful biomarkers of in vivo apoptosis. For example, serum levels of the caspase cleavage product of cytokeratin-18 have been used as a marker of chemotherapeutic efficacy in prostate, breast, and testicular cancers (Kramer et al., Br J Cancer, 2006, 94, 1592-8; Olofsson et al, Clin Cancer Res, 2007, 13, 3198-208; de Haas et al, Neoplasia, 2008, 10, 1041-8). Although apoptotic cells are typically cleared by phagocytes such as macrophages, it has been hypothesized that local clearance mechanisms are overloaded in cases of high cellular turnover and death, causing dying apoptotic cells to undergo secondary necrosis (Linder et al, Cancer Lett, 2004, 1, 1-9). While the plasma membrane remains intact during apoptosis, it is compromised and ruptured during secondary necrosis. Such secondary necrosis of dying tumor cells is consistent with the observation of what are normally intracellular components such as cytochrome c, DNA, nucleosomes, and cytokeratin-18 in the vasculature of cancer patients during chemotherapy (Beachy et al., Cancer Immunol Immunother, 2008, 57, 759-75).
A logical extension of these findings is that other caspase-derived neo-epitopes besides caspase-cleaved cytokeratin-18 are released into the vasculature of cancer patients undergoing chemotherapy. Such additional caspase-proteolyzed proteins may represent novel prognostic, diagnostic, or pharmacodynamic biomarkers of in vivo apoptosis, predicting likely patient outcome, indicating the most suitable therapeutic regimen, or serving as markers of therapeutic response. Because of tumor and patient heterogeneity, the clinical utility of single biomarker assays can be limited (Anderson et al., Mol Cell Proteomics, 2002, 1, 845-67). A multiparameter diagnostic assay of in vivo apoptosis based on a panel of caspase-derived neo-epitopes would likely be more sensitive and specific for a given type of cancer or therapeutic regimen. Great utility therefore exists in the identification of physiologically relevant caspase cleavage sites. Knowledge of such cleavage sites is required for the preparation of both peptide standards corresponding to neo-epitopes and antibodies that specifically bind to neo-epitopes. These reagents will enable identification and quantitation of caspase-derived neo-epitopes in biological samples such as serum, plasma, or tissue biopsies, and for validation of a given set of caspase-derived neo-epitopes as clinically useful biomarkers of in vivo apoptosis.